The work described here was carried out in collaboration with Javier Arroyo, of the Universidad Complutense in Madrid, Spain, and with Vladimir Farkas, of the Slovak Academy of Sciences, Bratislava, Slovakia. J. Arroyo provided strains and V. Farkas supplied oligosaccharides linked to sulforhodamine, which were synthesized in his laboratory. Work with those strains and with the oligosaccharides was carried out in its entirety in my laboratory.[unreadable] In previous periods, I studied, in collaboration with the laboratory of J. Arroyo, the function and regulation of Crh1p and Crh2p, two proteins that we found to be required for the linkage of chitin to beta(1-6)glucan in the budding yeast cell wall (1). From the nature of the postulated reaction and from homology of the Crh proteins with bacterial and plant enzymes, it was expected that these proteins would function as transglycosylases, transferring fragments of chitin chains to beta(1-6)glucan. To verify this hypothesis, it was desirable to obtain in vitro evidence for the reaction. However, an important obstacle to this endeavor is that the chitin chains, as available for in vitro experimentation, are hydrogen-bonded, therefore completely insoluble in water and probably not susceptible to transglycosylation. In vivo, the Crh proteins, at the plasma membrane or the cell wall, presumably have access to individual chitin chains emerging through the membrane and can act on them before they join other chains to form insoluble fibers. Many attempts to use in vitro soluble derivatives of chitin, such as carboxymethylchitin and glycolchitin, as substrates were unsuccessful. Therefore, in consultation with V. Farkas, I tried to develop a new approach. Saccharomyces cerevisiae was cultivated in the presence of fluorescent (sulforhodamine-linked) oligosaccharides, in the hope that they could penetrate the cell wall because of their small size, thus enabling the Crh proteins to use them as acceptors. This turned out to be the case. Cells grown in the presence of a mixture of beta(1-3)glucose oligosaccharides became fluorescent, whereas those grown with beta(1-6)glucose oligosaccharides did not. It should be noted that, as we found in a previous study, although in vivo there is chitin linked to ?(1-6)glucan, the point of attachment is not to the main beta(1-6) chain, but a branch glucose linked to it through a beta (1-3) bond. Thus, it makes sense that the oligosaccharides containing beta (1-3) linkages were the acceptors. The fluorescence extended all around the cell cortex, but was especially strong at bud scar locations, where previous cell divisions took place. It was not known that these areas would contain a greatly enhanced concentration of beta (1-6)glucan bound to chitin; however, this result coincides with the previous observation of a predominant concentration of Crh2p at the bud scar (2). Strains with deletions in both CRH1 and CRH2, as well as strains deleted for CHS3, which codes for the chitin synthase that produces the required chitin, exhibited a striking difference with wild type: the bright spots at bud scars were eliminated and the fluorescence around the cortex diminished. It was hypothesized that the remaining fluorescence may be due to Gas1p, a cell wall tranglycosylase that transfers a fragment of beta (1-3)glucan to another chain of the same polysaccharide, thereby elongating it. Conceivably, this enzyme could use the beta (1-3)-linked oligosaccharides as acceptors. Growth of a gas1? strain with the oligosaccharides resulted in a spectacular increase of fluorescence at the bud scars and also enhanced fluorescence around the cell cortex. This is in agreement with the known overexpression of Chs3p, Crh1p and Crh2p in this mutant. However, deletion of both CRH1 and CRH2 in this strain led to a total disappearance of the fluorescence, thus confirming that the Crh proteins are responsible for fluorescence at both sites.[unreadable] Tests with individual sulforhodamine-labeled oligosaccharides showed an increase in fluorescence with molecular weight, from the disaccharide to the heptasaccharide.[unreadable] In an attempt to get closer to a completely in vitro system, we made use of a 25-year old finding in our laboratory, that permeabilization of yeast cells with digitonin, while killing the cells, results in a strong activation of chitin synthases. Therefore, I decided to use the permeabilized cells as individual test tubes, by adding chitin synthase substrate (UDP-N-acetylglucosamine) and chitin acceptors (sulforhodamine oligosaccharides) to them, in the hope that the nascent chitin formed in the cells would be transferred to the acceptors. The experiment was successful. After fluorescent material unspecifically attached to the cells was eliminated by successive incubations with a protease and SDS, the cells were still fluorescent. In controls without UDP-N-acetylglucosamine the fluorescence was greatly reduced. A mutant overexpressing Crh2p showed a substantial increase in fluorescence, whereas a double mutant crh1delta crh2delta had no fluorescence at all.[unreadable] Taken together, all these results confirm the notion that Crh1p and Crh2p act as transglycosylases between chitin and its natural acceptor, beta (1-6)glucan, and point to an unexpected accumulation of the product at the site of cell division, which warrants further study.[unreadable] [unreadable] (1) Cabib, E., Blanco, N., Grau, C., Rodrguez-Pea, J.M., and Arroyo, J. (2007) Mol. Microbiol. 63, 921-935.[unreadable] (2) Rodrguez-Pea, J.M., Cid, V.J., Arroyo, J., and Nombela, C. (2000) Mol. Cell Biol. 20, 3245-3255.